Tetrahydrobiopterin (BH4) is the natural election donor for aromatic ring hydroxylases. Although it is a key component in the metabolism of phenylalanine and the formation of catecholamines and serotonin, its biosynthetic pathway is not totally resolved. The final step in the pathway from GTP to BH4 is controversial with some researchers claiming that dihydrobiopterin is reduced by Dihydrofolate reductase. To examine this, we used organ culture of rat pineal glands which contain high levels of BH4. Culture of rat pineal glands in methotrexate (0.5, 5 or 10 uM) for 6 or 24 h did not alter pineal tetrahydrobiopterin (85-90% of total biopterin in cultured glands), except for a small decrease of 30% after 24 h culture in 10 uM methotrexate. However, pineal dihydrobiopterin and/or biopterin (10-15% of total biopterin) was increased by methotrexate up to 2.5-fold. Biopterin detected in the culture medium following pineal culture was also increased to a similar extent after methotrexate and appeared to represent leakage of pineal dihydrobiopterin and/or biopterin. Culture of glands in 5 uM methotrexate did not alter the conversion of [U-14C] guanosine to [14C]biopterin, suggesting that pineal tetrahydrobiopterin synthesis was not altered by methotrexate. Complete inhibition of dihydrofolate reductase activity measured in pineal homogenates was obtained following culture of glands in all concentrations of methotrexate studied. Therefore, the enzyme dihydrofolate reducatase does not appear to be involved in a major biosynthetic pathway for pineal tetrahydrobiopterin from GTP, although it may have a minor role in tetrahydrobiopterin synthesis.